Establishment of micropropagation and cryopreservation protocols for the critically endangered medicinal plant, siphonochilus aethiopicus (Schweinf.) B.L. Burtt.

Abstract

In South Africa, more than 80% of rural communities rely on the trade of medicinally important plant species. Overexploitation coupled with biotic, abiotic, and climate change-induced stresses have reduced these wild populations. Since the Sustainable Development Goals adopted by the United Nations recognise the importance of protecting the planet’s natural resources to alleviate poverty and improve healthcare worldwide, increasing production and implementing ex situ long-term conservation strategies for these plant genetic resources is essential. The Zingiberaceae species Siphonochilus aethiopicus, a perennial geophyte, is amongst the top ten sought-after traditional medicinal plants in South Africa, which has led to its overexploitation and consequent critically endangered status. The anti-inflammatory activity of the plant’s major phytocompound, a furanoterpernoid called siphonochilone, from the rhizomes and roots has prompted further interest in this species by the pharmaceutical industry. Vegetative propagation via separated rhizome pieces containing buds is the only feasible method to grow this species due to poor seeding, which further complicates long-term storage through seed banking. Hence, an alternate long-term ex situ conservation strategy is needed for this species. Furthermore, established micropropagation protocols result in low yields and there are no reports on the siphonochilone content in the rhizomes and roots of regenerated plants. This study, therefore, aimed to develop conservation strategies for Siphonochilus aethiopicus through micropropagation and cryopreservation. A further aim was to validate and quantify siphonochilone in micropropagated clones and to compare this with conventionally propagated plants. During the establishment of in vitro cultures of S. aethiopicus, decontamination of rhizomederived bud explants proved challenging. A multi-step procedure incorporating a fungicide and a hypochlorite-based surface disinfectant yielded a small number of sterile buds. Thereafter, buds were cultured on full-strength Murashige and Skoog (MS) basal salts and vitamins, 0.09 M sucrose, 5 mg l-1 6-benzylaminopurine (BAP) and 4 g l-1 Gelrite® to promote development into shoots. Shoot apices were then excised and transferred to multiplication medium for 42 days. Multiplied shoots were elongated with a reduced BAP concentration (0.1 mg l-1) for 28 days. Elongated shoots were then rooted on medium containing 1 mg l-1 indole-3-acetic acid (IAA) for 28 days. Acclimatisation (42 days) was successful, with 100% survival. The plant yield in this study was much higher (13 plants per initial rhizome bud) than conventional vegetative propagation (a single plant per bud) or previously established micropropagation protocols (3 – 4 plants per bud). The mature two-year-old post-acclimatised micropropagated plants produced rhizomes (35.80 ± 6.11 g) and roots (15.70 ± 2.00 g) with higher biomasses compared to conventionally propagated plants (3.73 ± 2.48 g and 1.14 ± 0,22 g, respectively). Gas Chromatography-Mass Spectrometry (GC-MS) analyses identified siphonochilone in micropropagated and soil-grown plants. There were no significant differences in siphonochilone yield in rhizomes from the two-year-old micropropagated plants compared to that from field-cultivated plants. However, the root siphonochilone yield in the mature twoyear- old post-acclimatised micropropagated plants was higher (0.03120 ± 0.1109 to 0.4498 ± 0.1081 g g-1extract) than in roots of conventionally propagated plants (0.0435 ± 0.0136 g g- 1extract). To establish a cryopreservation protocol using in vitro-derived shoot apices, various cryopreparative and sequential dehydration procedures prior to cryostorage were evaluated. The shoot apices were precultured on sucrose-enriched medium and cryoprotected prior to vitrification and cryostorage. Shoot apices that were precultured on MS basal salt and vitamin medium with 0.09 M sucrose and 3 g l-1 Gelrite® for 48 h and then with 0.3 M sucrose in the dark for 24 h, followed by sequential dehydration, showed the highest viability and survival (58.33%) and a water content reduced to 0.92 ± 0.36 g g-1 dry weight of explants prior to cooling. The droplet-vitrification of the apices after this sequential dehydration process followed by cooling and storage in liquid nitrogen for 24 h resulted in 66.67% regrowth of the cryostored shoot apices following thawing, rehydration and recovery. Development of the shoot apex into a single shoot took place 90 days after retrieval from liquid nitrogen followed by elongation (28 days) and in vitro rooting (28 days). The plantlets generated from cryostored explants produced a single shoot and root, in contrast to untreated shoot apices (control) which produced robust shoots and roots in vitro. The successful recovery of cryostored explants represents the initial step towards long-term conservation of S. aethiopicus. Further research is recommended to improve explant recovery rates. Additionally, research is necessary to determine the presence and levels of siphonochilone in plants regenerated from cryostored explants. The improved micropropagation protocol for S. aethiopicus established in this study using in vitro-derived shoot apices, provide a viable recovery method for successful plant production following cryopreservation. Nonetheless, plant yields can be improved by optimising multiplication steps in temporary immersion culture systems, siphonochilone yield can be enhanced by investigating cell suspension cultures for phytocompound extraction, and optimisation of certain steps of the developed cryopreservation protocol can promote higher explant recovery.